Bottom Line:
The cytotoxic effect and the antiviral activity varied significantly within Eucalyptus species oils.In addition, E. odorata oil showed the most cytotoxic effect.However, the best antiviral activity appeared with E. bicostata.

Background: In 1957, Tunisia introduced 117 species of Eucalyptus; they have been used as fire wood, for the production of mine wood and to fight erosion. Actually, Eucalyptus essential oil is traditionally used to treat respiratory tract disorders such as pharyngitis, bronchitis, and sinusitis. A few investigations were reported on the biological activities of Eucalyptus oils worldwide. In Tunisia, our previous works conducted in 2010 and 2011 had been the first reports to study the antibacterial activities against reference strains. At that time it was not possible to evaluate their antimicrobial activities against clinical bacterial strains and other pathogens such as virus and fungi.

Methods: The essential oils of eight Eucalyptus species harvested from the Jbel Abderrahman, Korbous (North East Tunisia) and Souinet arboreta (North of Tunisia) were evaluated for their antimicrobial activities by disc diffusion and microbroth dilution methods against seven bacterial isolates: Haemophilus influenzae, Klebsiella pneumoniae, Pseudomonas aeruginosa, Staphylococcus aureus, Streptococcus agalactiae, Streptococcus pneumoniae and Streptococcus pyogenes. In addition, the bactericidal, fungicidal and the antiviral activities of the tested oils were carried out.

Results: Twenty five components were identified by GC/FID and GC/MS. These components were used to correlate with the biological activities of the tested oils. The chemical principal component analysis identified three groups, each of them constituted a chemotype. According to the values of zone diameter and percentage of the inhibition (zdi, % I, respectively), four groups and subgroups of bacterial strains and three groups of fungal strains were characterized by their sensitivity levels to Eucalyptus oils. The cytotoxic effect and the antiviral activity varied significantly within Eucalyptus species oils.

Mentions:
The essential oils were tested for their putative antibacterial activity against seven bacterial isolates represented by 81 strains (Table 2). As shown in Table 2, E. odorata oil possessed the best activity against S. aureus (27.4 ± 10.7 mm, zdi), followed by S. agalactiae (19.4 ± 5.6 mm, zdi), H. influenzae (19.2 ± 9.6 mm, zdi), S. pyogenes (19.0 ± 0.0 mm, zdi) and S. pneumoniae (17.4 ± 4.1 mm, zdi). E. maidenii oil showed a relatively good activity against S. aureus (22.8 ± 6.8 mm, zdi). To evaluate the correlation between the antibacterial activities and the essential oils of the eight Eucalyptus species, all the mean values of the zone diameters inhibition were subject to the PCA and the HCA analysis. The statistical analysis of the antibacterial activities of the oils showed a significant difference among Eucalyptus species oils and the tested bacterial strains (p < 0.05). The PCA horizontal axis explained 59.39% of the total variance, while the vertical axis a further 13.84% (Figure 3). The HCA showed two species groups (I and II), identified by their bacteria growth inhibition with a dissimilarity ≥ 15.0 (Figure 4). When the dissimilarity was ≥ 5.0, group II was divided into three subgroups (IIa, IIb and IIc). The horizontal axis permitted the separation of group I from group II, however axis II separated all the species of the group II into three subgroups. Group I, limited to the Gram negative (Gram(−)) bacteria, P. aeruginosa and K. pneumoniae, forms a deep dichotomy within the HCA analysis and a clearly separated group in the PCA analysis. These two strains were the most resistant to the majority of Eucalyptus essential oils with zdi < 7.1 mm for P. aeruginosa and 10.7 ± 1.5 mm for K. pneumoniae. Compared to the growth inhibition zone produced by ciprofloxacin against P. aeruginosa (34.7 ± 5.0 mm, zdi) and K. pneumoniae (32.4 ± 2.9 mm, zdi). Subgroup Ia was limited to S. aureus which was characterized by the highest sensitivity to E. maidenii and E. odorata oils (22.8 ± 6.8 and 27.4 ± 10.7 mm, zdi, respectively). This high sensitivity could be due to the disposition of E. maidenii and E. odorata oils with a relatively high mean percentage of the monoterpene hydrocarbons p-cymene (7.4 ± 2.9, 16.7 ± 5.2%, respectively). Previous studies have reported the high sensitive character of S. aureus to essential oils with a high content of p-cymene [35]. In addition, other researchers reported that this sensitivity of S. aureus was due to the single layer wall of the bacteria [36]. Comparing these results with those obtained with antibiotics, E. odorata essential oil produced a similar inhibition to that produced by gentamicin, erythromycin, vancomycin and benzylpenicillin (29.6 ± 6.2, 29.9 ± 5.1, 25.3 ± 4.4 and 24.5 ± 7.5 mm, dzi, respectively). However, this activity remained lower than that produced by fosfomycin (34.3 ± 11.1 mm, dzi). Sub group IIb represented by S. pneumoniae, showed a particular sensitivity to E. odorata and E. bicostata essential oils (17.4 ± 4.1 and 17.0 ± 4.0 mm, zdi). This inhibition remained lower than that produced by its specific antibiotics with zone diameters inhibition ranging from 26.3 ± 12.0 mm (erythromycin) to 35.6 ± 5.5 mm (fosfomycin). E. lehmannii, E. sideroxylon and E. cinerea oils did not show significant antibacterial activities with inhibition zones of 9.8 ± 2.4, 10.7 ± 2.5 and 11.5 ± 2.8 mm, respectively. Subgroup IIc, consists of Streptococcus B, S. pyogenes and H. influenzae. These strains were separated from all the others and correlated positively with the two axes and with E. cinerea and E. sideroxylon, the essential oils of which were characterized by a comparable activity against the previous bacterial strains, with inhibition zone diameters varying from 11.6 ± 1.4 mm to 13.0 ± 6.3 mm. Their activities were considered relatively as being lower than the tested antibiotics such as rifamicine and ampicilline. However E. odorata oil, which was removed from this group, showed the best activity against these bacterial strains with inhibition zone diameters varying from 17.4 ± 4.1 mm for S. pneumoniae to 19.4 ± 5.6 for Streptococcus B, but it remained much lower than that produced by their specific antibiotics. The MIC was performed for oils which have produced an inhibition ≥ 17 mm for clinical bacterial strains such as H. influenzae (reference 160), S. agalactiae (reference 3) S. pyogenes (reference 545) and S. aureus (reference 278). The result of their MIC was listed in Table 3. E. odorata and E. bicostata oils were characterized by the lowest MIC for Hemophylis influenzae (reference 160) (0.306 mg/mL), followed by S. agalactiae (reference 3) (10.4 mg/mL). These results were confirmed by the disc diffusion method. The highest MIC against S. aureus (reference 278) was shown for the oils of E. bicostata (169 mg/mL), E. odorata (156.6 mg/mL) and E. maidenii (151.8 mg/mL). This finding was in contradiction to results obtained by the disc diffusion method. According to the classification of Schaechter et al. (1999) [37] and Soro et al. (2010) [38], E. odorata, E. bicosta and E. maidenii oils were considered bactericidal (MBC/MIC < 4) against the tested strains, however the two first oils showed a better bactericidal activity against H. influenzae (reference 160) and S. aureus (reference 278) than that obtained with S. pyogens (reference 545) and S. aglatctiae (reference 3).

Mentions:
The essential oils were tested for their putative antibacterial activity against seven bacterial isolates represented by 81 strains (Table 2). As shown in Table 2, E. odorata oil possessed the best activity against S. aureus (27.4 ± 10.7 mm, zdi), followed by S. agalactiae (19.4 ± 5.6 mm, zdi), H. influenzae (19.2 ± 9.6 mm, zdi), S. pyogenes (19.0 ± 0.0 mm, zdi) and S. pneumoniae (17.4 ± 4.1 mm, zdi). E. maidenii oil showed a relatively good activity against S. aureus (22.8 ± 6.8 mm, zdi). To evaluate the correlation between the antibacterial activities and the essential oils of the eight Eucalyptus species, all the mean values of the zone diameters inhibition were subject to the PCA and the HCA analysis. The statistical analysis of the antibacterial activities of the oils showed a significant difference among Eucalyptus species oils and the tested bacterial strains (p < 0.05). The PCA horizontal axis explained 59.39% of the total variance, while the vertical axis a further 13.84% (Figure 3). The HCA showed two species groups (I and II), identified by their bacteria growth inhibition with a dissimilarity ≥ 15.0 (Figure 4). When the dissimilarity was ≥ 5.0, group II was divided into three subgroups (IIa, IIb and IIc). The horizontal axis permitted the separation of group I from group II, however axis II separated all the species of the group II into three subgroups. Group I, limited to the Gram negative (Gram(−)) bacteria, P. aeruginosa and K. pneumoniae, forms a deep dichotomy within the HCA analysis and a clearly separated group in the PCA analysis. These two strains were the most resistant to the majority of Eucalyptus essential oils with zdi < 7.1 mm for P. aeruginosa and 10.7 ± 1.5 mm for K. pneumoniae. Compared to the growth inhibition zone produced by ciprofloxacin against P. aeruginosa (34.7 ± 5.0 mm, zdi) and K. pneumoniae (32.4 ± 2.9 mm, zdi). Subgroup Ia was limited to S. aureus which was characterized by the highest sensitivity to E. maidenii and E. odorata oils (22.8 ± 6.8 and 27.4 ± 10.7 mm, zdi, respectively). This high sensitivity could be due to the disposition of E. maidenii and E. odorata oils with a relatively high mean percentage of the monoterpene hydrocarbons p-cymene (7.4 ± 2.9, 16.7 ± 5.2%, respectively). Previous studies have reported the high sensitive character of S. aureus to essential oils with a high content of p-cymene [35]. In addition, other researchers reported that this sensitivity of S. aureus was due to the single layer wall of the bacteria [36]. Comparing these results with those obtained with antibiotics, E. odorata essential oil produced a similar inhibition to that produced by gentamicin, erythromycin, vancomycin and benzylpenicillin (29.6 ± 6.2, 29.9 ± 5.1, 25.3 ± 4.4 and 24.5 ± 7.5 mm, dzi, respectively). However, this activity remained lower than that produced by fosfomycin (34.3 ± 11.1 mm, dzi). Sub group IIb represented by S. pneumoniae, showed a particular sensitivity to E. odorata and E. bicostata essential oils (17.4 ± 4.1 and 17.0 ± 4.0 mm, zdi). This inhibition remained lower than that produced by its specific antibiotics with zone diameters inhibition ranging from 26.3 ± 12.0 mm (erythromycin) to 35.6 ± 5.5 mm (fosfomycin). E. lehmannii, E. sideroxylon and E. cinerea oils did not show significant antibacterial activities with inhibition zones of 9.8 ± 2.4, 10.7 ± 2.5 and 11.5 ± 2.8 mm, respectively. Subgroup IIc, consists of Streptococcus B, S. pyogenes and H. influenzae. These strains were separated from all the others and correlated positively with the two axes and with E. cinerea and E. sideroxylon, the essential oils of which were characterized by a comparable activity against the previous bacterial strains, with inhibition zone diameters varying from 11.6 ± 1.4 mm to 13.0 ± 6.3 mm. Their activities were considered relatively as being lower than the tested antibiotics such as rifamicine and ampicilline. However E. odorata oil, which was removed from this group, showed the best activity against these bacterial strains with inhibition zone diameters varying from 17.4 ± 4.1 mm for S. pneumoniae to 19.4 ± 5.6 for Streptococcus B, but it remained much lower than that produced by their specific antibiotics. The MIC was performed for oils which have produced an inhibition ≥ 17 mm for clinical bacterial strains such as H. influenzae (reference 160), S. agalactiae (reference 3) S. pyogenes (reference 545) and S. aureus (reference 278). The result of their MIC was listed in Table 3. E. odorata and E. bicostata oils were characterized by the lowest MIC for Hemophylis influenzae (reference 160) (0.306 mg/mL), followed by S. agalactiae (reference 3) (10.4 mg/mL). These results were confirmed by the disc diffusion method. The highest MIC against S. aureus (reference 278) was shown for the oils of E. bicostata (169 mg/mL), E. odorata (156.6 mg/mL) and E. maidenii (151.8 mg/mL). This finding was in contradiction to results obtained by the disc diffusion method. According to the classification of Schaechter et al. (1999) [37] and Soro et al. (2010) [38], E. odorata, E. bicosta and E. maidenii oils were considered bactericidal (MBC/MIC < 4) against the tested strains, however the two first oils showed a better bactericidal activity against H. influenzae (reference 160) and S. aureus (reference 278) than that obtained with S. pyogens (reference 545) and S. aglatctiae (reference 3).

Bottom Line:
The cytotoxic effect and the antiviral activity varied significantly within Eucalyptus species oils.In addition, E. odorata oil showed the most cytotoxic effect.However, the best antiviral activity appeared with E. bicostata.

Background: In 1957, Tunisia introduced 117 species of Eucalyptus; they have been used as fire wood, for the production of mine wood and to fight erosion. Actually, Eucalyptus essential oil is traditionally used to treat respiratory tract disorders such as pharyngitis, bronchitis, and sinusitis. A few investigations were reported on the biological activities of Eucalyptus oils worldwide. In Tunisia, our previous works conducted in 2010 and 2011 had been the first reports to study the antibacterial activities against reference strains. At that time it was not possible to evaluate their antimicrobial activities against clinical bacterial strains and other pathogens such as virus and fungi.

Methods: The essential oils of eight Eucalyptus species harvested from the Jbel Abderrahman, Korbous (North East Tunisia) and Souinet arboreta (North of Tunisia) were evaluated for their antimicrobial activities by disc diffusion and microbroth dilution methods against seven bacterial isolates: Haemophilus influenzae, Klebsiella pneumoniae, Pseudomonas aeruginosa, Staphylococcus aureus, Streptococcus agalactiae, Streptococcus pneumoniae and Streptococcus pyogenes. In addition, the bactericidal, fungicidal and the antiviral activities of the tested oils were carried out.

Results: Twenty five components were identified by GC/FID and GC/MS. These components were used to correlate with the biological activities of the tested oils. The chemical principal component analysis identified three groups, each of them constituted a chemotype. According to the values of zone diameter and percentage of the inhibition (zdi, % I, respectively), four groups and subgroups of bacterial strains and three groups of fungal strains were characterized by their sensitivity levels to Eucalyptus oils. The cytotoxic effect and the antiviral activity varied significantly within Eucalyptus species oils.